Methods for producing macromolecule-identifying polymers

ABSTRACT

The methods for producing macromolecule identifying polymers according to the present invention comprise the steps of polymerizing a starting monomer in an aqueous solution in the presence of a macromolecule, a crosslinking agent, and a radical polymerization initiator to produce a polymer containing the macromolecule in its interior; and removing the macromolecule from the polymer containing the macromolecule to thereby produce the macromolecule identifying polymer having a molecular imprint of the macromolecule. In this method, the crosslinker has a solubility in water at 25° C. of 100% by mass or higher.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/JP04/002852, filed Mar. 5, 2004, which claims the benefit ofJapanese Patent Application No. 2003-070453, filed Mar. 14, 2003.

TECHNICAL FIELD

This invention relates to methods for producing polymers which arecapable of identifying macromolecules. More specifically, this inventionrelates to methods for producing polymers capable of identifyingmacromolecules in which polymerization of a starting monomer isconducted in an aqueous solution by using a particular crosslinker.

BACKGROUND ART

Recently, the process of analyzing, purifying, and detecting proteinsand other macromolecules has become a staple technology in the fields ofbiotechnology, drug discovery, therapeutics, clinical analysis, chemicalanalysis, and the like. Accordingly, materials and methods capable ofeffectively identifying a target protein or the like are highly awaited.

To date, to meet such a need, a method has been proposed for producing amolecularly imprinted polymer which has an imprint of a protein, inwhich the polymer is produced by radical polymerization of a monomer anda crosslinker in the presence of the protein (Non-patent document 1).This method, however, does not disclose any particular technique tosynthesize the polymer with optimal molecular identifying capacity for aparticular protein, and thus, trial and error experimentation isnecessary for the implementation of this method.

Most molecularly imprinted polymers are prepared in an organic solvent,such as acetonitrile or chloroform, and show selectivity for smallmolecules, such as sugars, amino acids, and steroids (Non-patentdocuments 2 to 4). More specifically, these methods use a small moleculeas template to synthesize a molecularly imprinted polymer in an organicsolvent. Accordingly, these methods cannot be applied to the synthesisof a molecularly imprinted polymer to be used for capturing awater-soluble macromolecule, such as a protein, wherein the synthesishas to be conducted in an aqueous solution.

Accordingly, many researchers are trying to synthesize a molecularlyimprinted polymer which can selectively identify a macromolecule in anaqueous solution.

For example, researchers have attempted to entrap a protein withinpolysiloxane or polyacrylamide in a buffer solution to thereby form themolecularly imprinted polymer (Non-patent Documents 5 to 8). Thisattempt, however, suffered from the problems of insufficientlyreproduced protein shape, insufficient strength and softness of thepolymer, and low selectivity. Another attempt was made with the use ofpolyaminophenylboronic acid to embed peroxidase in the disposablemicrotiter plate surface coating (Non-patent Document 9).

In a more recent attempt, a molecularly imprinted polymer wassynthesized by a method called “surface-imprinting procedure”, toselectively capture the protein (Non-patent Documents 10 and 11). Thismethod, involving the use of a metal (Cu²⁺) chelating monomer, however,could be applied only to proteins having the special structure withhistidine residue being exposed on the surface.

Another attempt involved the use of a cyclodextrin-based functionalmonomer for the formation of the polymer imprinted with a polypeptidemolecule in an aqueous solution (Non-patent Documents 12 and 13). Thismethod, however, is applicable only to low molecular weight compoundssuch as dipeptides (Phe-Phe).

Meanwhile, the inventors of the present invention have discovered anapproach called “epitope approach”. In this approach, a polymerimprinted with the shape of a lower molecular weight protein, which isdifferent from the target protein but whose main structure comprises apart of the target protein, is synthesized in an organic solvent, andthe target protein having the higher molecular weight is selectivelyentrapped by using this polymer (Patent Document 1).

[Patent Document 1] Unexamined Published Japanese Patent Application No.(JP-A) 2001-55399

[Non-patent Document 1] Angew. Chem., Int. Ed. Engl. 1995, vol. 34, pp.1812-1832

[Non-patent Document 2] Clin. Chem., 42(1996) 1506

[Non-patent Document 3] Nature, 361(1993), 645

[Non-patent Document 4] Proc. Nat. Acad. Sci., USA 92(1995) 4788

[Non-patent Document 5] Biochim. Biophys. Acta, 1250(1995) 126

[Non-patent Document 6] Biochim. Biophys. Res. Commun., 227(1996) 419

[Non-patent Document 7] Chromatographia, 44(1997) 227

[Non-patent Document 8] Chem. Lett., (1998) 731

[Non-patent Document 9] Anal. Chem., 73(2001) 5281

[Non-patent Document 10] J. Mol. Recogn., 8(1995) 35

[Non-patent Document 11] J. Amer. Chem. Soc., 123(2001) 2072

[Non-patent Document 12] J. Mol. Recogn., 11(1998) 94

[Non-patent Document 13] Anal. Chim. Acta, 435(2001) 25

DISCLOSURE OF THE INVENTION

The present invention has been made in view of such situation, and anobjective of the present invention is to provide a macromoleculeidentifying polymer which has highly replicated molecular imprints, andhence, high selectivity for a macromolecule such as a protein. Anotherobjective is to provide a method for producing such a macromoleculeidentifying polymer in an aqueous solution.

The inventors of the present invention conducted an extensive study tosolve the problem as described above, and found that performing thepolymerization reaction in an aqueous solution by using a particularcrosslinker results in a molecule identifying polymer having highlyreplicated molecular imprints, and hence, high selectivity for thetarget macromolecule. The present invention has been completed on thebasis of such a finding.

The summary of the present invention is described below.

The method for producing a macromolecule identifying polymer accordingto the present invention comprises the steps of polymerizing a startingmonomer in an aqueous solution in the presence of a macromolecule, acrosslinker, and a radical polymerization initiator to produce a polymercontaining the macromolecule in its interior; and removing themacromolecule from the polymer containing the macromolecule to therebyproduce the macromolecule identifying polymer having a molecular imprintof the macromolecule. In this method, the crosslinker has a watersolubility at 25° C. of 100% by mass or higher.

Preferably, the crosslinker is polyethyleneglycol di(meth)acrylate.

Preferably, the polyethyleneglycol di(meth)acrylate has a number averagemolecular weight of 400 or more.

Preferably, the crosslinker is used at 1 to 200 moles per mole of thestarting monomer.

Preferably, the radical polymerization initiator is a water-soluble azocompound.

Preferably, the radical polymerization initiator has a 10 hour half-lifedecomposition temperature in the range of 30 to 50° C.

Preferably, the radical polymerization initiator is an azo compoundrepresented by the following general formula (I):

wherein R¹ and R² are independently a hydrogen atom or an alkyl groupcontaining 1 to 3 carbon atoms and may be the same or different; or asalt thereof.

The macromolecule used is a polypeptide, a polynucleotide, a sugar, or aderivative thereof.

Preferably, the polypeptide is one comprising 3 to 5000 amino acids or aderivative thereof.

Preferably, the starting monomer is a vinyl monomer.

The macromolecule identifying polymer according to the present inventionhas a molecular imprint of the macromolecule, and comprises structuralunits derived from a vinyl monomer, and a structural unit derived frompolyethyleneglycol di(meth)acrylate having a solubility in water at 25°C. of 100% by mass or higher. This macromolecule identifying polymerchanges its volume by 5% or less when it is immersed in water.

Preferably, the structural units derived from a vinyl monomer and thestructural unit derived from polyethyleneglycol di(meth)acrylate areobtained as a result of the polymerization at a ratio of 1 to 200 molesof said polyethyleneglycol di(meth)acrylate to 1 mole of said vinylmonomer.

The macromolecule identifying film according to the present inventioncomprises the macromolecule identifying polymer as described above.

The macromolecule identifying beads according to the present inventioncomprise the macromolecule identifying polymer as described above.

The method for screening a macromolecule according to the presentinvention is accomplished by bringing a plurality of macromolecules intocontact with the macromolecule identifying film or the macromoleculeidentifying bead of the present invention, under the conditions in whichthe target macromolecule can bind to such macromolecule identifying filmor beads.

Next, the method for producing a macromolecule identifying polymeraccording to the present invention is described in detail.

The method for producing the polymer capable of identifying amacromolecule (also referred herein to as the “macromolecule identifyingpolymer”) according to the present invention comprises the steps ofpolymerizing a starting monomer in an aqueous solution in the presenceof a macromolecule, a crosslinker, and a radical polymerizationinitiator to produce a polymer containing the macromolecule in itsinterior; and removing the macromolecule from the polymer containing themacromolecule to thereby produce the polymer having a molecular imprintof the macromolecule. The crosslinker has a solubility in water at 25°C. of 100% by mass or higher. The phrase “a crosslinker has a solubilityin water of 100% by mass” means that 100 parts by mass of thecrosslinker will dissolve in 100 parts by mass of water. This alsoapplies to the following description. As used herein, (meth)acrylateincludes methacrylate and acrylate.

Macromolecule

Macromolecules which may be used in the present invention include apolynucleotide, a polypeptide, and a sugar.

The polynucleotide used preferably comprises 3 to 5000 nucleotides. Thenucleotide may be modified with other functional groups, such as afluorescent label or isotope label.

Examples of such polynucleotides include double stranded DNA, singlestranded DNA, and RNA.

The polypeptide used preferably comprises 3 to 5000 amino acids, morepreferably 5 to 5000 amino acids, and most preferably 8 to 5000 aminoacids. The polypeptide may be modified with a sugar chain or otherfunctional group.

Such polypeptides are not particularly limited, and exemplarypolypeptides include various proteins, such as an enzymatic protein, abacterial protein, a microbial protein, an antigenic protein, a proteinof animal or vegetable origin, and other biological proteins as well asvarious synthetic proteins.

Exemplary sugars used in the present invention include a monosaccharide,a disaccharide, an oligosaccharide (including a trisaccharide andtetrasaccharide), or a polysaccharide, and examples include glucose,mannose, fructose, ribose, xylose, arabinose, galactose, aldonic acid,glucitol, maltose, cellobiose, lactose, sucrose, trehalose, andmaltotriose.

The term “polysaccharide” as used herein refers to sugar in a broadsense, and includes alginic acid, cyclodextrin, cellulose, and othersubstances that are generally found in the nature. The derivatives ofsuch sugar include reducing sugars of such sugar, for example, a sugaralcohol [as represented by general formula: HOCH₂(CHOH)_(n)CH₂OH(wherein n is an integer of 2 to 5)] and oxidized sugars of such sugar,for example, an aldonic acid or a uronic acid.

Starting Monomer

The matrix of the macromolecule identifying polymer produced by theproduction method of the present invention is polymerized from astarting monomer, which is not particularly limited for its type so longas it is soluble in water. Examples of such starting monomers include amonomer having vinyl group (vinyl monomer) and a monomer havingvinylidene group.

Examples of such vinyl monomers include water-soluble organic compoundshaving one or more vinyl groups, for example, (meth)acrylic acid, itsalkaline metal salt, styrene sulfonate, its alkaline metal salt,(meth)acrylamide, N,N-dimethylaminopropylacrylamide, andN,N-dimethylacrylamide. The metal used in the alkaline metal salt ispreferably sodium or potassium, and more preferably, sodium.

Of such vinyl monomers, the preferred are N,N-dimethylacrylamide andmetal acrylates, such as sodium acrylate, and the most preferred aremetal acrylates, such as sodium acrylate.

Use of such a vinyl monomer enables the introduction of a functionalgroup from the vinyl monomer into the molecular imprint in themacromolecule identifying polymer. This enables the production of amacromolecule identifying polymer with improved selectivity, owing tothe synergetic effects of the interaction with the macromolecule and themolecular imprint.

The vinyl monomer as described above may be used either alone or incombinations of two or more.

The amount of the starting monomer used depends on the type ofmacromolecule used, and is not particularly limited. The startingmonomer, however, is preferably used at an amount of 1 mole or more,more preferably 3 moles or more, and most preferably 4 moles or more permole of the macromolecule. With regard to the upper limit, the startingmonomer is preferably used at 100 moles or less, more preferably at 50moles or less, and most preferably at 20 moles or less per mole of themacromolecule.

Use of the starting monomer in such a range enables the production of amacromolecule identifying polymer having an improved selectivity.

Crosslinker

The crosslinker used in the present invention has a solubility in waterat 25° C. of 100% by mass or higher, preferably 300% by mass or more,and most preferably infinite. The term “infinite” is used for thesolubility when the crosslinker dissolves in water to form a homogeneousmixture, and such homogeneous state is maintained irrespective ofincrease in the amount of the crosslinker added.

Examples of such crosslinkers include polyethyleneglycoldi(meth)acrylate.

Such polyethyleneglycol di(meth)acrylate preferably has a number averagemolecular weight of at least 400, more preferably 400 to 1000, and stillmore preferably 500 to 800.

Use of polyethyleneglycol di(meth)acrylate is particularly preferablewhen the radical polymerization initiator described below is a compoundrepresented by formula (I).

Such a crosslinker may be used either alone or in combinations of twoore more, and optionally, in combination with other crosslinkers, solong as the objective of the present invention is not adverselyaffected.

Use of polyethyleneglycol di(meth)acrylate as the crosslinker enablespolymerization of the starting monomer in an aqueous solution at a highdegree of crosslinking. This, in turn, enables precise replication ofthe macromolecule to the imprint, as well as production of amacromolecule identifying polymer having a high strength which does notexperience collapse of the imprint. Such a macromolecule identifyingpolymer exhibits high selectivity.

The crosslinker is used in the present invention at an amount thatallows for polymerization. For example, the crosslinker is preferablyused with water at an amount (volume ratio of water to the crosslinker)in the range of 99:1 to 1:99, and more preferably 70:30 to 1:99.

The crosslinker is preferably used at an amount of, for example, 1 moleor more, more preferably 2 moles or more, still more preferably 5 molesor more, and most preferably 10 moles or more per mole of the startingmonomer. With regard to the upper limit, the crosslinker is preferablyused at 200 moles or less, more preferably at 100 moles or less, stillmore preferably at 60 moles or less, and most preferably at 40 moles orless per mole of the starting monomer.

Use of polyethyleneglycol di(meth)acrylate in such an amount enablespolymerization of the starting monomer in an aqueous solution at a highdegree of crosslinking. This, in turn, enables precise replication ofthe macromolecule to the imprint, as well as production of amacromolecule identifying polymer having a high strength which does notexperience collapse of the imprint. Such a macromolecule identifyingpolymer exhibits high selectivity.

Since the polymer is strong and free from imprint collapse, decrease inthe selectivity by repeated use of the macromolecule identifying polymercan be avoided.

Conventional macromolecule identifying polymers polymerized in anaqueous solution are formed on a substrate, such as glass, since thepolymer is insufficient in its strength. Use of the crosslinkeraccording to the present invention eliminates the necessity of such asubstrate for polymer formation.

Radical Polymerization Initiator

The radical polymerization initiator used in the present invention ispreferably a water-soluble azo compound.

Of the water-soluble azo compounds, preferred is a radicalpolymerization initiator having a 10 hour half-life decompositiontemperature of 30° C. to 50° C., more preferably 35° C. to 45° C.Herein, the phrase “10 hour half-life decomposition temperature” refersto the temperature at which the amount of the radical polymerizationinitiator becomes ½ of its initial amount in 10 hours when it is heatedto such temperature in an aqueous solution and retained at suchtemperature.

As described above, the radical polymerization initiator used in thepresent invention is preferably soluble in water and reacts at a lowtemperature as described above.

Such a radical polymerization initiator is preferably an azo compoundrepresented by the following formula (I):

or a salt thereof.

In the formula (I), R¹ and R² are a hydrogen atom or an alkyl groupcontaining 1 to 3 carbon atoms. Exemplary alkyl groups include methyl,ethyl, n-propyl, and isopropyl.

R¹ and R² may be the same or different, but are preferably the same.

Of the compounds as described above, the preferred is the one wherein R¹and R² are both a hydrogen atom or a methyl group.

The salts of the azo compound represented by formula (I) are preferablyacidic salts, such as a hydrochloride or a sulfate.

Examples of such radical polymerization initiators include:

2,2′-azobis[2-(2-imidazoline-2-yl)propane]dihydrochloride, representedby the formula:

(10 hour half-life decomposition temperature: 44° C.);

2,2′-azobis[2-(2-imidazoline-2-yl)propane]disulfate dihydraterepresented by the formula:

(10 hour half-life decomposition temperature: 46° C.); and

2,2′-azobis[2-(5-methyl-2-imidazoline-2-yl)propane]dihydrochloriderepresented by the formula:

(10 hour half-life decomposition temperature: 41° C.).

Among these, the preferred is2,2′-azobis[2-(2-imidazoline-2-yl)propane]dihydrochloride having a 10hour half-life decomposition temperature of 44° C.

The amount of the radical polymerization initiator used may varyaccording to the polymerization temperature, the type of radicalpolymerization initiator, and the degree of contaminant oxygen presentduring the radical polymerization; for example, the amount may rangefrom 10⁻⁷ moles to 1 mole per mole of crosslinker. The preferred amountused may also be expressed in terms of the time required for completionof the polymerization; for example, the radical polymerization initiatoris preferably used approximately at an amount such that thepolymerization is completed within 200 hours, more preferably, within 80hours.

Such a radical polymerization initiator may be used alone or incombinations of two or more.

Use of such a radical polymerization initiator enables efficientpolymerization of the starting monomer as well as production at animproved efficiency of a highly crosslinked macromolecule identifyingpolymer with high strength. The resulting macromolecule identifyingpolymer also has improved selectivity.

Aqueous Solution

The macromolecule identifying polymer according to the present inventionis synthesized by polymerization in an aqueous solution. Exemplaryaqueous solutions include distilled water, purified water, ultrapurewater, various salines, and pH buffer solutions such as phosphatesolutions. When the aqueous solution is a saline or pH bufferingsolution, the solution preferably has the lowest possible concentrationof each salt, at the level of up to several ten mM. When the aqueoussolution is a pH buffer solution, the pH is adjusted so as to avoiddenaturing the macromolecule, such as a polynucleotide, protein, orsugar.

Such an aqueous solution preferably has a pH of 3 to 10 to preventdenaturing of the macromolecule, such as a protein, and, in the case ofprotein, the aqueous solution is preferably maintained at a pH near theisoelectric point of the protein.

The aqueous solution used in the present invention may also have anorganic solvent added thereto, so long as the organic solvent does notinterfere with the activity or conformation of the macromolecule.Exemplary organic solvents include trifluoroethanol,hexafluoroisopropanol, acetonitrile, and dimethylsulfoxide.

When an organic solvent is added, it is preferably used at an amount inthe range of 1 to 100 parts by volume per 100 parts by volume of theaqueous solution, although the amount may vary depending on the type ofthe organic solvent used.

Addition of the organic solvent within a certain range will suppresschanges in the conformation of the macromolecule during thepolymerization.

Other Additives

In the production of the macromolecule identifying polymer of thepresent invention, other additives may also be added, so long as suchaddition does not adversely affect the objective of the presentinvention. Examples of such other additives include a polymerizationaccelerator (e.g., N,N,N′,N′-tetramethylenediamine), diluent,polymerization inhibitor, plasticizer, UV absorbent, antioxidant,antistatic, anti-mold agent, moisture adjusting agent, and flameretardant, which may be used either alone or in combinations of two ormore.

Substrate

The molecule identifying polymer produced by the production method ofthe molecule identifying polymer according to the present invention ishighly crosslinked, and hence, has a high strength. Accordingly, it canretain its shape without using any substrate, and can be used as is. Themolecule identifying polymer, however, may also be used by forming thepolymer on the substrate.

In such a case, the substrate used is preferably either an inorganicsolid substance or an organic solid substance, and it may be eitherporous or nonporous.

Examples of inorganic substrates include silica gel, alumina, titania,zirconia, silica-alumina, zeolite, glass, and gold, among which glassand gold are more preferred.

Preferred organic substrates include beads of a cured resin, such asbeads of melamine resin.

The substrate may take the shape of powders, particles, plates, andvarious other shapes.

Such substrate is preferably one which has been surface treated in orderto improve binding strength between the substrate and the polymer of thestarting monomer.

For example, when the substrate used is glass, the surface treatingagent preferably has vinyl group bonded thereto. The substrate having avinyl group on its surface can be produced by surface treating thesubstrate with a silane coupling agent having a vinyl group, oralternatively, by reacting the substrate having a functional group, suchas an amino group, having active hydrogen on its surface with a reactivevinyl compound, such as acryloyl chloride. When such substrate having avinyl group is used, the polymer film formed on the substrate will reactwith and bind to this vinyl group, and, thus, the polymer will be firmlybonded to the substrate.

When the substrate used is gold, a vinyl group can be introduced to thegold surface by bringing an organic compound which has a vinyl group andwhich also has an —SH group or —S—S bond for covalent bonding to thegold surface (for example, N,N′-bis(acryloyl)-cystamine) in contact withthe gold.

Method for Producing a Macromolecule Identifying Polymer

The method for producing the macromolecule identifying polymer accordingto the present invention comprises the steps of polymerizing thestarting monomer in the aqueous solution in the presence of themacromolecule, the crosslinker, and the radical polymerization initiatorto produce a polymer containing the macromolecule in its interior; andremoving the macromolecule from the polymer containing the macromoleculeto thereby produce a polymer having a molecular imprint of themacromolecule. In this case, the crosslinker has a solubility in waterat 25° C. of 100% by mass or higher.

The polymerization may be accomplished, for example, by adding thestarting monomer and the crosslinker to an aqueous solution containingthe macromolecule, bubbling an inert gas, such as nitrogen gas, into themixture solution to purge oxygen, and adding a polymerization initiatorand optionally a polymerization accelerator and the like to the mixtureto thereby allow the polymerization to take place.

The polymerization is preferably conducted at room temperature (approx.25° C.) to 50° C., and more preferably at a temperature in the range of35 to 40° C., preferably for approximately 1 to 200 hours. When thepolymerization temperature is lower than the above-specified range, thepolymerization will be insufficient, or the formation of themacromolecule imprint may take place while the gel is swollen to detractfrom the high precision replication. When the polymerization temperatureis higher than such range, problems may arise, such as denaturing of thetarget protein and the resulting undesired imprint of the denaturedprotein, and, in such a case, the resulting polymer may be incapable ofidentifying the target non-denatured macromolecule.

When the macromolecule identifying polymer is to be formed on asubstrate, the polymerization may be allowed to take place on thesubstrate to thereby form a polymer film containing the protein or othermacromolecule on the substrate.

In the method for producing a macromolecule identifying polymeraccording to the present invention, the macromolecule is removed fromthe thus formed polymer containing the macromolecule in its interior tothereby form a void at the site of the macromolecule removal, such voidfunctioning as a keyhole for the target macromolecule.

More specifically, for example, when the macromolecule identifyingpolymer is formed in the form of beads (particles), the polymercontaining the macromolecule produced by the polymerization ispulverized and washed with an aqueous solution or the like to therebyremove the macromolecule that had been used in forming the imprint.

When the macromolecule identifying polymer is produced in the form of afilm, the aqueous solution containing the components, including thestarting monomer, is polymerized on a planar substrate to thereby form afilm containing the macromolecule in its interior. The film is thenwashed with an aqueous solution or the like to thereby remove themacromolecule that had been used in forming the imprint.

The aqueous solution used for the washing may be any one of distilledwater, purified water, ultrapure water, various salines, pH buffersolutions such as phosphate solutions, and the like, as in the case ofthe solvent used in dissolving the macromolecule and the startingmonomer.

In order to facilitate the removal of the macromolecule, the washingsolution may further contain, for example, a protein denaturant, such asurea or guanidine hydrochloride, or a surfactant, such as sodium dodecylsulfate, dodecylpyridinium chloride, or octylglycoside. When suchdenaturant or surfactant is used, the polymer is preferably furtherwashed after the washing of the protein or the like to thereby removethe denaturant or the surfactant; exemplary solutions which may be usedin this step include distilled water, purified water, ultrapure water,various salines, and pH buffer solutions such as phosphate solution.

The thus produced macromolecule identifying polymer may be stored byimmersing in distilled water or the like.

Macromolecule Identifying Polymer and its Application

The macromolecule identifying polymer according to the present inventionis a macromolecule identifying polymer having a molecular imprint of themacromolecule comprising, for example, structural units derived from thevinyl monomer, and structural unit derived from the polyethyleneglycoldi(meth)acrylate, having a solubility in water at 25° C. of 100% by massor higher.

With regard to the content of the structural units derived from thevinyl monomer and the structural unit derived from thepolyethyleneglycol di(meth)acrylate, the structural unit derived fromthe polyethyleneglycol di(meth)acrylate is preferably incorporated at1.5 moles or more, more preferably at 2 moles or more, still morepreferably at 5 moles or more, and most preferably at 10 moles or moreper mole of the structural units derived from the vinyl monomer. Withregard to the upper limit in the content of the structural unit derivedfrom the polyethyleneglycol di(meth)acrylate, this unit is preferablyincorporated, for example, at 200 moles or less, more preferably at 100moles or less, still more preferably at 60 moles or less, and mostpreferably at 40 moles or less per mole of the structural units derivedfrom the vinyl monomer.

The structural units derived from the vinyl monomer and the structuralunit derived from the polyethyleneglycol di(meth)acrylate are the mainconstituents of the macromolecule identifying polymer, and thesecomponents are preferably incorporated in the macromolecule identifyingpolymer at a total content of 50% by mole or more, more preferably at90% by mole or more, and most preferably at 99% by mole or more.Exemplary other components include a monomer different from thosedescribed above, a radical polymerization initiator, and otheradditives.

The macromolecule identifying polymer according to the present inventionas described above will enjoy a high degree of crosslinking andexcellent strength. It is also substantially free from swelling by waterabsorption or the like or contraction by drying as well as deformationof the shape. Also, the macromolecule identifying polymer of the presentinvention experiences only a slight decrease in selectivity afterrepeated use.

The macromolecule identifying polymer preferably changes its volumeafter water immersion (for 24 hours or more) by 5% or less, preferably3% or less, and more preferably 1% or less. The minimum value of thevolume change is preferably 0%. The volume change of the macromoleculeidentifying polymer after drying the polymer that had been immersed inwater is also preferably within equivalent range.

The macromolecule identifying polymer may be formed into any desiredshapes including films and beads (particles).

The thickness of such film is not limited, and may vary according to itsapplication. The thickness, however, is preferably in the range of 1 to100 μm.

When the polymer is used in the form of beads, the average particle sizeis not limited, and may vary according to its application. The averageparticle size, however, is preferably in the range of 1 to 100 μm.

The macromolecule identifying polymer of the present invention hasimprints of the macromolecule replicated at a remarkably high fidelityas well as high polymer strength with little risk of imprintdeformation; therefore, it can be used in the form of a film withoutusing a substrate.

The macromolecule identifying polymer of the present invention may beused in screening for (separating) the target macromolecule from amixture solution containing the target macromolecule, such as a protein.Accordingly, the macromolecule identifying polymer of the presentinvention can be used in the screening, for example, as a sensor fordetecting the particular macromolecule, such as protein, or in place ofan antibody when used in an assay or measurement system that had beenrealized by the use of such an antibody. More specifically, themacromolecule identifying polymer in the shape of a film can be used asa molecule identifying chip, and the macromolecule identifying polymerin the shape of beads can be used as a filler for columns.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described in further detail below withreference to Examples, but should not be construed as being limitedthereto.

EXAMPLE 1

To 2.06 mL of distilled water was dissolved 25 μmol (23.1 mg) of [Sar¹,Ala⁸]-angiotensin II (manufactured by American Peptide; amino acidsequence: Sar-Arg-Val -Tyr-Ile-His-Pro-Ala, hereinafter also referred toas “SA”). To this solution were then added 100 μmol (10 μL) ofN,N-dimethylacrylamide (manufactured by Wako Pure Chemical Industries,Ltd., hereinafter also referred to as “DMAAm”), 5 mmol (2.565 mL) ofpolyethyleneglycol diacrylate (manufactured by Aldrich, Mn=575;solubility, infinite), and 515 μL (50 mM) of sodium phosphate buffersolution (pH 7.2), and then, about 100 μL of NaOH aqueous solution (0.5M) to adjust the pH to 7.1.

Nitrogen gas was introduced to this mixture for 30 seconds to removeoxygen. To this mixture was then added 26 μmol of2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044(product name) manufactured by Wako Pure Chemical Industries, Ltd.;hereinafter also referred to as “ABIPD”; 10 hour half-life decompositiontemperature, 44° C. (in water)).

The mixture was then purged with nitrogen gas for 2 minutes, and thetemperature was maintained at 37° C. for 60 hours to obtain the polymer.

The resulting polymer was pulverized in mortar, and sieved to separateand collect the polymer having the particle size in the range of 0.02 to0.045 mm. The polymer was washed 5 times with 20 mM phosphate buffersolution (10 mL, pH 5.6), and the supernatant of the solution used forwashing was confirmed so that it did not contain the SA used to form theimprint. The macromolecule identifying polymer (1) having the SA removedtherefrom was thereby produced.

COMPARATIVE PREPARATION EXAMPLE 1

The procedure of Example 1 was repeated, with the exception that the SAused for the imprint formation was not added in this case to therebyproduce Reference polymer (1).

EXAMPLE 2

To 1.17 mL of distilled water was dissolved 20 μmol (18.5 mg) of SA. Tothis solution were then added 80 μmol (40 μL of 2 M aqueous solution) ofsodium acrylate (manufactured by Aldrich), 3.5 mmol (1.80 mL) ofpolyethyleneglycol diacrylate (manufactured by Aldrich; Mn=575), 3 mmol(0.70 mL) of polyethyleneglycol diacrylate (manufactured by Aldrich;Mn=258; solubility, infinite), and 1250 μL (20 mM) sodium phosphatebuffer solution (pH 7.2), and then, about 100 μL of NaOH aqueoussolution (0.5 M) to adjust the pH to 7.1.

Nitrogen gas was introduced to this mixture for 30 seconds to removeoxygen. To this mixture was then added 26 μmol of ABIPD.

The mixture was then purged with nitrogen gas for 2 minutes, and thetemperature was maintained at 37° C. for 60 hours to obtain the polymer.

The resulting polymer was pulverized in mortar, and sieved to separateand collect the polymer having the particle size in the range of 0.02 to0.045 mm. The polymer was washed 5 times with 20 mM phosphate buffersolution (10 mL, pH 5.6), and the supernatant of the solution used forwashing was confirmed so that it did not contain the SA used to form theimprint. The macromolecule identifying polymer (2) having the SA removedtherefrom was thereby produced.

COMPARATIVE PREPARATION EXAMPLE 2

The procedure of Example 2 was repeated, with the exception that the SAused for the imprint formation was not added in this case to therebyproduce Reference polymer (2).

EXAMPLE 3

The procedure of Example 2 was repeated, with the exception that thesodium acrylate was used at an amount of 160 μmol (80 μL of 2 M aqueoussolution); 4 mmol (2.052 mL) of polyethyleneglycol diacrylate(manufactured by Aldrich; Mn=575) was used instead of the combined useof the polyethyleneglycol diacrylate (Mn=575) and the polyethyleneglycoldiacrylate (Mn=258); and the ABIPD was used at an amount of 26 μmol tothereby produce macromolecule identifying polymer (3).

COMPARATIVE PREPARATION EXAMPLE 3

The procedure of Example 3 was repeated, with the exception that the SAused for the imprint formation was not added in this case to therebyproduce Reference polymer (3).

EXAMPLE 4

The procedure of Example 3 was repeated, with the exception that thesodium acrylate was used at an amount of 320 μmol (160 μL of 2 M aqueoussolution) to thereby produce macromolecule identifying polymer (4).

COMPARATIVE PREPARATION EXAMPLE 4

The procedure of Example 4 was repeated, with the exception that the SAused for the imprint formation was not added in this case to therebyproduce Reference polymer (4).

Evaluation of Example 1

The particulate macromolecule identifying polymer (1) produced inExample 1 and the particulate Reference polymer (1) produced inComparative Preparation Example 1 were filled in a stainless steelcolumn (inner diameter, 4.6 mm; length, 10 cm) for liquidchromatography, respectively. A mixture of phosphate buffer solution(80% by volume) and acetonitrile (20% by volume) was prepared and thismobile phase was applied to the column at a column temperature of 25° C.and at a rate of 0.5 mL per min. to thereby conduct a separation assayexperiment of the SA and the comparative angiotensin II (manufactured byAmerican Peptide; amino acid sequence: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe;hereinafter also referred to as “All”) and Gly-Leu-Tyr (manufactured bySigma; hereinafter also referred to as “GLY”).

The protein identifying performance of the macromolecule identifyingpolymer (1) and Reference polymer (1) was evaluated by a monitoringmethod at 215 nm using a UV detector.

The results are shown in Table 1.

In the results shown in Table 1, the value I which indicates the degreeof formation of the imprint in the macromolecule identifying polymer(1), is 1.24 (0.56/0.45). This confirms that the molecular imprint ofthe SA was formed in the macromolecule identifying polymer (1) ofPreparation Example 1.

The value I indicates a difference in the separation ability for SAbetween the macromolecule identifying polymer (1) having the imprint ofSA and the Reference polymer (1) having no imprint; the largerdifference between the value I and 1 indicates the successful formationof the SA imprint.

The value of the separation coefficient, α, which indicates selectivityof the macromolecule identifying polymer (1) for SA was 2.80(0.56/0.20). This confirms that this macromolecule identifying polymer(1) has the function of identifying the AII.

The value α indicates the ability of the macromolecule identifyingpolymer (1) to separate SA from AII in the use of the macromoleculeidentifying polymer (1), namely, selectivity for SA of the macromoleculeidentifying polymer (1) in the relative comparison of SA and AII, andthe larger difference between the value α and 1 indicates the higherselectivity.

Evaluation of Examples 2 to 4

Separation assay experiments between the SA and the comparative AII andGLY were conducted for the macromolecule identifying polymers (2) to (4)produced in Examples 2 to 4 by repeating the evaluation procedure ofExample 1 using the corresponding Reference polymers (2) to (4). Theresults are also shown in Table 1.

TABLE 1 Comp. Comp. Comp. Comp. Ex. 1 Prep. Ex. 1 Ex. 2 Prep. Ex. 2 Ex.3 Prep. Ex. 3 Ex. 4 Prep. Ex. 4 SA (μmol) 25 0 20 0 20 0 20 0 DMAAm 100100 — — — — — — (μmol) Sodium — — 80 80 160 160 320 320 acrylate (μmol)PEGDA-575 5 5 3.5 3.5 4 4 4 4 (mmol) PEGDA-258 — — 3.0 3.0 — — — —(mmol) ABIPD (μmol) 26 26 26 26 26 26 26 26 k(SA) 0.56 0.45 4.25 3.143.19 1.66 6.63 4.33 k(AII) 0.20 0.11 0.16 0.15 0.11 0.12 0.14 0.08k(GLY) 0.55 0.43 0.28 0.36 0.48 0.42 0.44 0.36 I 1.24 — 1.35 — 1.92 —1.53 — α(SA/AII) 2.80 — 26.56 — 28.25 — 47.40 — α(SA/GLY) 1.02 — 15.18 —6.59 — 15.10 — SA: [Sar¹, Ala⁸]-angiotensin II AII: Angiotensin II GLY:Gly-Leu-Tyr DMAAm: N,N-dimethylacrylamide PEGDA-575: polyethyleneglycoldiacrylate (Mn = 575) PEGDA-258: polyethyleneglycol diacrylate (Mn =258) ABIPD: 2,2′-azobis[2-(2-imidazoline-2-yl)propane]dihydrochloridek(SA): Capacity coefficient of SA {Retention time (min.) of SA in thecolumn chromatography using a column filled with the given polymer −retention time (min.) of the void marker (acetone)}/{retention time(min.) of the void marker} k(AII): Capacity coefficient of AII{Retention time (min.) of AII in the column chromatography using acolumn filled with the given polymer − retention time (min.) of the voidmarker (acetone)}/{retention time (min.) of the void marker} k(GLY):Capacity coefficient of GLY {Retention time (min.) of GLY in the columnchromatography using a column filled with the given polymer − retentiontime (min.) of the void marker (acetone)}/{retention time (min.) of thevoid marker} I: Imprint effect (k(SA) of the macromolecule identifyingpolymer/k(SA) of the reference polymer) α(SA/AII): Separationcoefficient (k(SA) of the macromolecule identifying polymer/ k(AII) ofthe macromolecule identifying polymer) α(SA/GLY): Separation coefficient(k(SA) of the macromolecule identifying polymer/ k(GLY) of themacromolecule identifying polymer)

EXAMPLE 5

To 1.634 mL of distilled water was dissolved 20 μmol (18.8 mg) of SA. Tothis solution were then added 200 μmol (100 μL of 2 M aqueous solution)of acrylic acid (manufactured by Aldrich), 2 mmol (1.026 mL) ofpolyethyleneglycol diacrylate (manufactured by Aldrich; Mn=575;solubility, infinite), 2 mmol (0.46 mL) of polyethyleneglycol diacrylate(manufactured by Aldrich; Mn=258; solubility, infinite), and 515 μL (50mM) of sodium phosphate buffer solution (pH 7.2), and then, about 100 μLof NaOH aqueous solution (0.5 M) to adjust the pH to 7.1.

Nitrogen gas was introduced to this mixture for 30 seconds to removeoxygen. To this mixture was then added 72 μL (26 μmol) of ABIPD.

The mixture was then purged with nitrogen gas for 2 minutes, and thetemperature was maintained at 37° C. for 60 hours to obtain the polymer.

The resulting polymer was pulverized in mortar, and sieved to separateand collect the polymer having the particle size in the range of 0.02 to0.045 mm. The polymer was washed 5 times with 240 mM aqueous solution ofsodium dihydrogenphosphate (7 mL, pH 5.6) and twice with 12 mM aqueoussolution of sodium dihydrogenphosphate (7 mL, pH 5.6), and thesupernatant of the solution used for washing was confirmed so that itdid not contain the SA used to form the imprint. The macromoleculeidentifying polymer (5) having the SA removed therefrom was therebyproduced.

COMPARATIVE PREPARATION EXAMPLE 5

The procedure of Example 5 was repeated, with the exception that the SAused for the imprint formation was not added in this case to therebyproduce Reference polymer (5).

COMPARATIVE EXAMPLE 1

The procedure of Example 5 was repeated, with the exception that acombination of 44 μmol of ammonium persulfate (manufactured by Wako PureChemical Industries, Ltd.) and 34 μmol of tetramethylethylenediamine(manufactured by Wako Pure Chemical Industries, Ltd., hereinafter alsoreferred to as “TEMED”) was used instead of the 26 μmol of ABIPD tothereby prepare Comparative polymer (1).

COMPARATIVE PREPARATION EXAMPLE 6

The procedure of Comparative Example 1 was repeated, with the exceptionthat the SA used for the imprint formation was not added in this case tothereby produce Reference polymer (6).

COMPARATIVE EXAMPLE 2

The procedure of Example 5 was repeated, with the exception that 19 μmolof azobismethoxyvaleronitrile (manufactured by Wako Pure ChemicalIndustries, Ltd., hereinafter also referred to as “ABMODV”) was usedinstead of the 26 μmol of ABIPD to thereby Comparative polymer (2).

COMPARATIVE PREPARATION EXAMPLE 7

The procedure of Comparative Example 2 was repeated, with the exceptionthat the SA used for the imprint formation was not added in this case tothereby produce Reference polymer (7).

Evaluation of Example 5

Separation assay experiments between the SA and the comparative GLY wereconducted for the macromolecule identifying polymer (5) produced inExample 5 by repeating the evaluation procedure of Example 1 using theReference polymer (5). The results are shown in Table 2.

Evaluation of Comparative Examples 1 and 2

Separation assay experiments between the SA and the comparative GLY wereconducted for the Comparative polymers (1) and (2) produced inComparative Examples 1 and 2 by repeating the evaluation procedure ofExample 1 using the corresponding Reference polymers (6) and (7). Theresults are also shown in Table 2.

TABLE 2 Comp. Comp. Comp. Comp. Comp. Ex. 5 Prep. Ex. 5 Ex. 1 Prep. Ex.6 Ex. 2 Prep. Ex. 7 SA (μmol) 20 0 25 0 44 0 Acrylic acid 200 200 200200 350 350 (μmol) PEGDA-258 2 2 2 2 2 2 (mmol) PEGDA-575 2 2 2 2 2 2(mmol) ABIPD (μmol) 26 26 — — — — Ammonium — — 44 + 34 44 + 34 — —persulfate (μmol) + TEMED (μmol) ABMODV — — — — 19 19 (μmol) k(SA) 2.961.43 0.93 1.25 0.90 0.96 k(GLY) 0.38 0.38 0.78 1.04 0.24 0.28 I 2.07 —0.74 — 0.94 — α(SA/GLY) 8.13 — 1.19 — 3.75 — SA: [Sar¹,Ala⁸]-angiotensin II GLY: Gly-Leu-Tyr PEGDA-575: polyethyleneglycoldiacrylate (Mn = 575) PEGDA-258: polyethyleneglycol diacrylate (Mn =258) ABIPD: 2,2′-azobis[2-(2-imidazoline-2-yl)propane]dihydrochlorideTEMED: tetramethylethylenediamine k(SA): Capacity coefficient of SA{Retention time (min.) of SA in the column chromatography using a columnfilled with the given polymer − retention time (min.) of the void marker(acetone)}/{retention time (min.) of the void marker} k(GLY): Capacitycoefficient of GLY {Retention time (min.) of GLY in the columnchromatography using a column filled with the given polymer − retentiontime (min.) of the void marker (acetone)}/{retention time (min.) of thevoid marker} I: Imprint effect (k(SA) of the macromolecule identifyingpolymer/k(SA) of the reference polymer) α(SA/GLY): Separationcoefficient (k(SA) of the macromolecule identifying polymer/k(GLY) ofthe macromolecule identifying polymer)[Evaluation of the Volume Change]

One polymer particle having an average diameter of 67.97 μm (Polymerparticle (A)) and one polymer particle having an average diameter of38.63 μm (Polymer particle (B)) were collected from the macromoleculeidentifying polymer (3) produced in Example 3 which had been immersed inwater for at least one day. The collection was carried out under themicroscope (magnification, 400).

These polymer particles were air dried at room temperature, and changein the average diameter over time was evaluated (at 0 min., 30 min., 120min., and 1000 min.). The average diameter was determined by measuringthe diameter of the polymer particle in three directions under themicroscope and calculating the average.

The results are shown in Table 3. As demonstrated in Table 3, nosubstantial change in the average diameter could be confirmed for eitherof the polymer particles (A) and (B) at 1,000 minutes, namely, after thedrying of the polymer particles. This indicates that the polymerparticles produced by the method of the present invention are highlycrosslinked and that the imprints are reliably formed.

TABLE 3 Average diameter (μm) Average diameter (μm) Time (min.) of thepolymer particle (A) of the polymer particle (B) 0 67.97 38.63 30 67.7138.66 120 67.31 38.82 1000 67.46 38.10

INDUSTRIAL APPLICABILITY

The macromolecule identifying polymer of the present invention isproduced by using a particular crosslinker for the polymerization, andtherefore, it has highly reproduced imprints of the targetmacromolecule, and hence, high selectivity for the target macromolecule.

1. A macromolecule identifying polymer having a molecular imprint of amacromolecule, comprising a structural unit derived from a vinyl monomerselected from the group consisting of N,N′-dimethylacrylamide, sodiumacrylate, and acrylic acid, and a structural unit derived frompolyethyleneglycol diacrylate having a solubility in water at 25° C. of100% by mass or higher, wherein said macromolecule identifying polymerchanges its volume by 5% or less when it is immersed in water; whereinsaid macromolecule identifying polymer is obtained by a methodcomprising polymerizing the vinyl monomer in an aqueous solution in thepresence of the macromolecule, polyethyleneglycol diacrylate and aradical polymerization initiator to produce a polymer containing themacromolecule in its interior, and removing the macromolecule from thepolymer containing the macromolecule to thereby produce themacromolecule identifying polymer having a molecular imprint of themacromolecule; and wherein said radical polymerization initiator is anazo compound represented by general formula (I)

wherein R¹ and R² are independently a hydrogen atom or an alkyl groupcontaining 1 to 3 carbon atoms and may be the same or different fromeach other, or a salt thereof.
 2. The macromolecule identifying polymeraccording to claim 1, wherein said structural unit derived from a vinylmonomer and said structural unit derived from polyethyleneglycoldiacrylate are obtained as a result of the polymerization at a ratio of1 to 200 moles of said polyethyleneglycol diacrylate to 1 mole of saidvinyl monomer.
 3. A macromolecule identifying film comprising themacromolecule identifying polymer of claim 1 or
 2. 4. A macromoleculeidentifying bead comprising the macromolecule identifying polymer ofclaim 1 or
 2. 5. The macromolecule identifying polymer of claim 1,wherein the radical polymerization initiator is:


6. The macromolecule identifying polymer of claim 5, wherein the radicalpolymerization initiator is


7. The macromolecule identifying polymer of claim 1, wherein thepolymerization is carried out at a temperature of 25-50° C.
 8. Themacromolecule identifying polymer of claim 1, wherein the polymerizationis carried out at a temperature of 35-40° C.
 9. The macromoleculeidentifying polymer of claim 1, wherein the polyethyleneglycoldiacrylate has a number average molecular weight of 400 or more.
 10. Themacromolecule identifying polymer of claim 1, wherein said macromoleculeis a polypeptide, a polynucleotide, a sugar, or a derivative thereof.11. A method for producing a macromolecule identifying polymer of claim1, comprising the steps of polymerizing a starting monomer in an aqueoussolution in the presence of a macromolecule, a crosslinker, and aradical polymerization initiator to produce a polymer containing themacromolecule in its interior, and removing the macromolecule from thepolymer containing the macromolecule to thereby produce a macromoleculeidentifying polymer having a molecular imprint of the macromolecule, andwherein said crosslinker has a solubility in water at 25° C. of 100% bymass or higher.
 12. The method according to claim 1, wherein thecrosslinker is polyethyleneglycol diacrylate having a number averagemolecular weight of 400 or more.
 13. The method according to claim 11,wherein said crosslinker is used at 1 to 200 moles per mole of saidstarting monomer.
 14. The method according to claim 11, wherein saidradical polymerization initiator has a 10 hour half-life decompositiontemperature in the range of 30 to 50° C.
 15. The method according toclaim 11, wherein said macromolecule is a polypeptide, a polynucleotide,a sugar, or a derivative thereof.
 16. The method according to claim 15,wherein said polypeptide comprises 3 to 5000 amino acids or a derivativethereof.